An efficient ELISA protocol for measurement of SARS-CoV-2 spike-specific IgG in human plasma and serum samples

Here, we describe a protocol for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA). The protocol was developed with a keen focus on optimizing several key parameters, including antigen coating concentration, antibody and sample dilutions, and assay development time. The final protocol features the following characteristics:• The capability to detect SARS-CoV-2 spike-specific IgG in both plasma and serum samples.• A streamlined procedure that requires only 1 hour and 20 minutes of hands-on time.• Reliable assay performance, with a remarkable sensitivity of 98.1 % and specificity of 99.5 %.


a b s t r a c t
Here, we describe a protocol for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA).The protocol was developed with a keen focus on optimizing several key parameters, including antigen coating concentration, antibody and sample dilutions, and assay development time.The final protocol features the following characteristics: • The capability to detect SARS-CoV-2 spike-specific IgG in both plasma and serum samples.
• A streamlined procedure that requires only 1 hour and 20 minutes of hands-on time.
• Reliable assay performance, with a remarkable sensitivity of 98.1 % and specificity of 99.5 %.

Subject area
Immunology and Microbiology More specific subject area Diagnostics Name of your method SARS-CoV-2 Spike-Specific IgG ELISA Name and reference of original method n.a.

Resource availability
Reagents, equipment, consumables, software

Reagents
Commercially available

Introduction
In 2019, the novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the widespread coronavirus disease 2019 (COVID-19) pandemic [1] .At the beginning of the pandemic, laboratory resources, including tests to measure SARS-CoV-2 antibody (Ab) responses, became limited and were primarily allocated to facilities involved in the immediate management of the health crisis [2] .To enable us to carry out SARS-CoV-2 research specifically relevant for human immunodeficiency virus (HIV) diagnostic and the community of individuals living with an HIV infection [3] , we developed an enzymelinked immunosorbent assay (ELISA) protocol for the detection of SARS-CoV-2 spike specific immunoglobulin G (IgG) in serum or plasma samples.In a nutshell, the protocol involves coating 96-well plates with SARS-CoV-2 spike protein in sodium hydrogen carbonate on the first day, followed by overnight incubation at 4 °C.Subsequently, the plates are washed and blocked for one hour at room temperature.After diluting the samples, they are added to the plate and incubated for a duration of 2 hours.Afterwards, the plate is washed, and an horseradish peroxidase (HRP)-labeled detection Ab specific to the crystallizable fragment (Fc) region of human IgG is added, followed by another one-hour incubation period.The plates are washed again, and 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB)-substrate is added for development, which is stopped using sulfuric acid.

Procedure
It is imperative for all experimenters to adhere to all applying regulations concerning necessary precautions, personal protective equipment, and laboratory sample handling.The ELISA should be conducted at room temperature, unless stated otherwise, and without any shaking.Throughout the incubation periods, it is essential to seal the plate with a plate sealer.To optimize efficiency, use a multichannel pipette and a reservoir whenever feasible.Similarly, for time-saving purposes, prepare sample dilutions in cell culture plates rather than 1.5 ml reaction vessels.

Day 1
Antigen immobilization 1. Prepare a 5 μg/mL final concentration of SARS-CoV-2 spike protein by diluting it in a 0.1 M sodium hydrogen carbonate solution (pH 8.6). 2. Add 100 μl of the diluted protein per well of a 96-well ELISA plate.3. Incubate the plate overnight at 4 °C.

Blocking of ELISA plates
If an automated plate washer is not available, you can use a multichannel pipette for plate washing.In that case, remove the washing buffer by flicking the plate after each wash and gently dry the plate on a paper towel before proceeding.
4. Wash the ELISA plate 4 times with 400 μl of PBS-T5 using an automated plate washer and blot it dry. 5. Add 360 μl PBSA-T1 per well.6. Incubate the plate for 1 h at room temperature.

Addition of samples
When handling human clinical samples (steps 7, 9, and 11), use a biosafety cabinet.For a project involving serum and plasma samples from SARS-CoV-2 convalescent individuals, we found that a dilution of 1:200 provides the best sample resolution.
7. During step 6, vortex the samples and dilute in PBSA-S in a 96-well cell culture plate.8. Wash the ELISA plate 4 times with PBS-T5 and blot it dry.9. Add 100 μl of sample dilution to each well of the ELISA plate.10.Incubate the ELISA plate for 2 h at room temperature.

Addition of detection Ab
11. Wash the ELISA plate 6 times with PBS-T5 and blot it dry.12. Briefly mix the detection Ab by vortexing and dilute 1:20,000 in PBSA.13.Add 100 μL of the diluted detection Ab to each well.14.Incubate for 1 h at room temperature.

Development and measurement
15. Wash the ELISA plate 6 times with PBS-T5 and blot it dry.16.Develop by adding 100 μL of 1-Step Ultra-TMB substrate.17.Incubate the plate in the dark for 30 min at room temperature.18. Stop the reaction by adding 100 μl of 1 N sulfuric acid to each well.19.Read the absorbance of each well at 450 nm with reference wavelength 570 nm using a plate reader.

Time taken
The execution of the ELISA according to the protocol typically takes approximately 5 hours and 30 minutes (excluding overnight incubation), with a hands-on time of around 1 hour and 20 minutes.These estimates assume that preparations for subsequent steps are carried out concurrently with ongoing incubations.

Procedure
Step Hands-on time Incubation time

Anticipated results
The SparkControl software of the Tecan Spark plate reader will generate an excel sheet containing the measurement results.The plate reader accurately detects optical density (OD) values between 0 and 4. In cases where the signal exceeds the detection limit, the plate reader displays "over" instead of a specific value.The ELISA assay readings are specifically measured at 450 nm, and it is essential for all assay values to fall within the designated detection range.If any values surpass the upper limit, it is necessary to repeat the ELISA with higher dilutions of the samples to prevent signal saturation.To determine the background signal of the assay plate, the reference wavelength of 570 nm is measured.It is crucial for these reference values to remain below 0.01.After subtraction of the reference wavelength signal from the sample signals, assay results are analyzed by GraphPad Prism 9. To ensure the validity of the assay, certain criteria must be met: a strong positive control should yield an OD of ≥ 1.5, while the negative control should not exceed an OD of ≤ 0.3.A sample is considered positive for SARS-CoV-2 spike IgG if its value exceeds the negative control signal by at least two-fold.

Troubleshooting
If the quality of the assay does not meet the expectations, please refer to the following suggestions regarding common sources of errors that may occur during the conduction of ELISAs.

Method validation
In order to develop the ELISA protocol, we systematically evaluated and adjusted several key assay parameters, including the coating concentration of soluble SARS-CoV-2 spike, assay development time, and dilutions of the detection Ab and human plasma samples.Initially, the draft protocol employed a coating concentration of 10 g/ml of SARS-CoV-2 spike, a 10-minute assay development time, and a detection Ab dilution of 1:40,000.While these conditions already allowed for the distinction of SARS-CoV-2 spike reactive and non-reactive human plasma samples, low sample dilutions of 1:80 or less were required to separate a sample with medium reactivity from the negative control ( Fig. 1A ).
To improve the ELISA protocol, essential assay parameters were subsequently optimized, and improved conditions were implemented as protocol modifications for subsequent refinements.First, we performed a titration experiment using 0.3-10 g/ml of SARS-CoV-2 spike and a sample dilution of 1:100 ( Fig. 1B ).This experiment demonstrated that a coating concentration of 5 g/ml

Fig. 1 .
Fig. 1.Characterization and optimization of an in-house ELISA protocol for the detection of SARS-CoV-2 spike-specific IgG (A) Results of the initial non-optimized ELISA protocol for detecting SARS-CoV-2 spike-specific IgG Abs in human plasma samples.(B) SARS-CoV-2 spike protein concentrations in the ELISA coating buffer were systematically varied.(C) Results of ELISA development times of 10, 20, and 30 minutes are shown.(D) Different dilutions of the detection Ab in the ELISA protocol were examined.Raw values exceeding the detection limit of the plate reader were labeled as "OVER".(E) Depiction of the sample resolution for a detection Ab dilution of 1:20,000.(F) Matching plasma and serum samples of 9 SARS-CoV-2 spike IgG + individuals were analyzed by ELISA.Potential differences between groups were determined by a two-sided paired t-test (  = 0.05).ns = non-significant.(G) Human plasma samples of individuals (n = 52) with diagnostically confirmed SARS-CoV-2 spike-specific IgG Abs were tested using the optimized in-house ELISA protocol to determine assay sensitivity.The data from all reactive samples in the in-house ELISA (n = 51) were plotted against the results of the in-vitro diagnostics certified Euroimmun anti-SARS-CoV-2 ELISA (IgG).(H) To evaluate the specificity of the in-house ELISA, serum samples (n = 185) collected prior to the corona pandemic in 2018 were analyzed.The dashed line represents the cutoff of 2-fold over background, above which samples are considered reactive.All assays were performed in duplicates or triplicates with at least two independent repetitions.All graphs show the grand mean.Any error bars depict the standard error of the mean.Pictures were generated using GraphPad Prism 9.